1. Field of the Invention
The present invention relates to a reagent sheet for use in the measurement of .gamma.-glutamyl transferase activity in a liquid sample and an integral multilayer analytical element for use in the measurement of said activity, and more particularly to a reagent sheet and an integral multilayer analytical element for use in the analysis of aqueous sample solutions, particularly in the analysis of .gamma.-glutamyl transferase activity in body fluids in clinical test.
2. Description of Prior Arts
It is very important to measure the activity of .gamma.-glutamyl transferase in human body fluids in clinical test. As enzyme activity relating to a cancer of the liver, bile duct or the pancreas, there is frequently done the measurement of the activity of .gamma.-glutamyl transferase (hereinafter referred to sometimes as GGT) in body fluids in the diagnosis of said cancer.
As the method of measurement of GGT activity, there had been reported a GGT activity measuring method using a synthetic substrate by A. Szewczuk M. Orlowski in 1960 [see, Clin. Chem. Acta, 5; 680-688, (1980)]. Since then, various synthetic compounds for the measurement of GGT activity had been synthesized, systems of conjugation reactions with color-forming reactions had been developed, and various methods are widely used at the present time [see, Japanese Patent Provisional Publication Nos. 54(1979)-133191, 53(1978)-111793, 54(1979)-132533, 56(1981)-148298, 56(1981)-148299, 56(1981)-164796, 56(1981)-23897, 56(1981)-92845, 56(1981)-158745, 56(1981)-169597, 56(1981)-30956, 53(1978)-147034, 54(1979)-28195 and 55(1980)-26870].
Most of the aforementioned methods use consecutive reactions in which pH of the reaction system is changed to form a color or there is added a color reagent which has a fear of said reagent itself or an intermediate inhibiting an enzyme reaction. Though there are only a few cases at present, there are synthesized some substrates which allows the enzyme reaction itself to be directly observed, that is, some self-developable substrates having velocity method applicability.
As the self-developable substrates for the measurement of GGT activity, there are known .gamma.-glutamyl-p-nitroanilide developed by M. Orlowski [see, Arch. Immunol. Ther. Exp., 13, 538 (1985)] and .gamma.-glutamyl-3-carboxy-4-nitroanilide disclosed in Japanese Patent Publication No. 54(1979-7781.
The latter is superior in solubility and used as a substrate for IFCC standard method. Although the former is poor in solubility, it has been early developed and its manufacturing cost is low, so that the substrate has been most studied, its clinical knowledge is rich, and it is widely used presently. In an analytical method using .gamma.-glutamyl-p-nitroanilide as self-developable substrate, there is used a surfactant for dissolving said .gamma.-glutamyl-p-nitroanilide which is difficultly soluble. However, it is known that the use of a cationic or anionic surfactant alone inhibits GGT activity. Thus, in conventional analytical methods, there is a serious problem how to dissolve .gamma.-glutamyl-p-nitroanilide without inhibiting GGT activity.
As a method for dissolving .gamma.-glutamyl-p-nitroanilide without inhibiting GGT activity, there is disclosed in Japanese Patent Publication No. 57(1982)-24759 a method in which .gamma.-glutamyl-p-nitroanilide is dissolved in an aqueous solution containing both a cationic surfactant and a nonionic surfactant. Further, Japanese Patent Provisional Publication No. 55(1980)-153600 discloses a freeze-dried agent which can be almost instantaneously dissolved in a bufferred test solution at 20.degree. to 25.degree. C. by adding a stoichiometric or excess amount of anionic surfactant to a base equal amount of .gamma.-glutamyl-p-nitroanilide to form a protonized substrate and freeze-drying it. Conventional analytical methods including the above-described methods are those which are a so-called wet analytical method in which an analyte reacts with a reagent in a diluted aqueous solution.
An analytical method which can be performed with only a small amount of a sample and can be easily operated with high accuracy, has been highly desired by medical persons such as medical doctors in clinical test. Therefore, GGT activity-measuring methods using a dry analytical method in place of said conventional wet analytical method have been developed in recent years. In the dry analytical methods, reagents required for detection reaction are preserved in a dry state and the reaction of these reagents with a small quantity of a sample solution is detected to make the measurement. Among the dry analytical methods, there have been developed analytical methods using an integral multilayer analytical element which is easy to handle and the improvements of the element are being made. The integral multilayer analytical element for the measurement of GGT activity comprises a porous spreading layer containing a substrate which is self-developable in response to contact with .gamma.-glutamyl-p-nitroanilide, a support layer and optionally other intervening functional layers such as a water-absorbing layer prepared by laminating them for integration.
The dry analytical methods using such an integral multilayer analytical element was reported by H. G. Curme, et al. [Clim. Chem., 24, 1335-1350 (1978) and B. Walter [Analytical Chemistry, (1983) 55, 493A].
However, when the activity of .gamma.-glutamyl transferase is measured by these wet analytical methods, there are caused other problems in addition to those associated with the conventional wet analytical methods in which .gamma.-glutamyl-p-nitroanilide is poor in solubility and the surfactants inhibits GGT activity. Unlike the wet analytical method, the substrate and other reagents are preserved in dry state in the dry analytical method. Therefore, when the substrate preserved in a dry state is brought into contact with a small amount of water in a liquid sample, the substrate must be readily re-dissolved in order to improve the accuracy of the measurement of GGT activity and to make the measurement over a wide range.